Hox transcriptional regulators play important roles in mammalian development. While their role in the development and regeneration of skin and its appendages is currently understudied, this is receiving increasing attention. A notable case is Hoxc13, which is essential for normal hair follicle development. Both hoxc/3-overexpressing transgenic mice created by us and Hoxc13 null mice exhibit severe hair growth defects resulting in alopecia. DMA microarray analysis of the transgenic mice and subsequent validation by various methods identified Foxql and Foxnl (mutated in nude and satin), proto-oncogene Junb, desmosomal cadherin gene Dsc2, and a several hair keratin genes as putative targets of Hoxc13 regulation. Our results support the hypothesis that Hoxc13 plays a central role in a complex regulatory network controlling hair follicle differentiation. Defining this network is critical for understanding mechanisms involved in numerous skin and hair diseases, including diseases that result in alopecia and some skin cancers. Our overall aims are to 1) determine whether Hoxc13 directly interacts with specific target genes. Control elements will be defined by DNA binding, co-transfection and chromatin immunoprecipitation assays, and Hoxc13-dependent regulation in vivo using reporter gene analysis in both transgenic and null mice. 2) We will determine whether Hoxc13 and Foxgl proteins regulate putative common target genes either cooperatively or independently. We will test functionality of putative Foxql and Hoxc13 binding sites in the promoter region of Dsc2 as a paradigm in vitro, and determine whether these mediate potential cooperative/synergistic responses in co-transfection assays. 3) Lastly, we will define Hoxc13-dependent regulatory gene networks essential for hair follicle differentiation. Comparative longitudinal DNA microarray analysis will be done using skin samples from both transgenic and null mice with appropriate controls at 5 time points. These data will define the effect of over- or nonexpression of Hoxc13 at distinct morphogenetic stages of hair follicle development. Use of Ingenuity Network Analysis(c) and other bioinformatics tools will allow us to develop a conceptual framework of a Hoxc13-controlled genetic network regulating hair follicle differentiation. Relevance to Public Health: We are defining a gene network that controls development of hair follicles and hair shafts in mammals. Mutations in any or all of these genes result in a various types of alopecia, many of which are identical to specific human skin diseases. Our goal is to determine how these genes interact with each other, which will ultimately define how these diseases can be approached therapeutically.